1. Field of the Invention
The present invention relates to carrier supports such as chromatographic supports, and methods for their preparation and use. More particularly, the invention relates to carrier supports comprising a polymeric carrier grafted to a substrate for affinity chromatography media and for the affinity chromatography media made from these supports. The invention also relates to chromatographic devices which permit radial or tangential flow of a sample relative to the carrier support.
2. Brief Description of the Background Art
In the technique of affinity chromatography, which enables the efficient isolation of biological macromolecules or biopolymers by utilizing their recognition sites for certain supported chemical structures with a high degree of selectivity, the prior art has utilized materials of varying chemical structure as supports. For example, agarose gels and cross-linked agarose gels have been the most widely used support materials. Their hydrophilicity makes them relatively free of nonspecific binding, but their compressibility makes them less attractive as carriers in large scale processing, such as in manufacturing. Controlled-pore glass (CPG) beads have also been used in affinity chromatography. Although high throughputs can be obtained with columns packed with CPG, this carrier is even more expensive than agarose beads. Cellulose particles have also been used by immunochemists for synthetic affinity sorbents. However, compared to agarose gels, cellulose particles are formed with more difficulty and therefore, have received less attention in the preparation of affinity sorbents for enzymes. Cellulose, however, is perhaps the least expensive of all support matrices. Two lesser-used support matrices are polyacrylamide gel beads and Sephadex .RTM. gel beads made from dextran and epichlorohydrin. Although convenient methods have been developed for using them, the softness of these beads yields poor column packings, and their low molecular porosity yields a sorbent with poor ligand availability to the ligate.
Coupek et al., U.S. Pat. No. 4,281,233, shows supports for affinity chromatography which comprise copolymers of hydroxy alkyl acrylates or methacrylates with cross-linking monomers. The copolymers contain covalently attached mono- or oligosaccharides. (An oligosaccharide is defined in the art as having up to nine saccharide units. See, e.g., Roberts, J. D., and Caserio, M. C., Basic Principles of Organic Chemistry, 1964, p. 615.)
A carrier for bio-active materials is also disclosed in Nakashima et al., U.S. Pat. No. 4,352,884. The Nakashima carrier comprises a substrate coated with a copolymer. The substrate may be one of various materials, including inorganic materials such as glass; silica; alumina; synthetic high polymers such as polystyrene, polyethylene, and the like; and naturally occurring high polymers such as cellulose. The copolymer is made of a hydrophilic acrylate or methacrylate monomer which is a hydroxy or alkoxyalkylacrylate or methacrylate, and a copolymerizable unsaturated carboxylic acid or amine. The base material or substrate is coated with the copolymer by conventional coating or deposition procedures, such as spraying, dipping, phase separation or the like. The copolymer may also contain small amounts of a cross-linking agent such as glycidyl acrylate or methacrylate. The cross-linking agent allows for cross-linking treatment after the coating process, and provides for the prevention of elution (presumably of the bioactive materials) from the coating layer. The amounts of cross-linking agent are quite small, and range between 0.5 and 1% by weight of the total copolymer weight. Such amounts of cross-linking agent are insufficient to cause substantial covalent bonding or grafting of the copolymer onto the underlying substrate. The copolymer in Nakashima is thus essentially only physically coating the underlying substrate. Physical coating, however, is accompanied by a series of problems. The carrier would not be expected to have an even distribution of the copolymer, would show a multilayered structure, and may have a possible uneven distribution of functional groups.
Another reference of interest is Kraemer, U.S. Pat. No. 4,070,348, which shows copolymers of glycidyl- and amino-containing acrylates, useful as carriers for biologically active substances, such as polysaccharides, enzymes, peptides, hormones, etc. The structure of the final product in Kraemer is that of an acrylic copolymer chain covalently modified at a multiplicity of sites thereon with substances such as enzymes, proteins, and the like.
This review of the prior art, its advantages and drawbacks, leads to the conclusion that there exists a need for a support useful both for ion exchange and affinity chromatography-based purification which will have high stability, high porosity, low non-specific adsorption, high flow rate, non-compressibility, controlled gelation, and which will be useful for industrial-scale biological separations. It is at the industrial level of manufacturing, especially, where the aforementioned drawbacks of the prior art have had their most important effect and where this need is the strongest.
Industrial-scale molecular separation materials comprising fibrous matrices with particulate immobilized therein have been described in commonly assigned U.S. Pat. No. 4,384,957 to Crowder, which is herein incorporated by reference. This patent describes a composite fiber material formed by wet laying a sheet from an aqueous slurry of particulate, small refined fiber pulp, and long soft fiber pulp.
Using a fibrous/particulate matrix with addition of cationic polymers to the slurry and cross-linking the polymers to the matrices by oven-drying has yielded a filtration matrix with a positive charge coated on the surface thereof. Such charged matrices can be used for filtering minute amounts of particulate impurities from large volumes of liquid by adsorption. (See, for example, Ostreicher, U.S. Pat. Nos. 4,007,113 and 4,007,114, as well as U.S. Pat. Nos. 4,305,782 and 4,309,247, which are all herein incorporated by reference.)
It is inevitable in prior art wet slurrying processes with slurries comprising cationic materials to obtain materials having uneven distribution of charges, wherein multilayer coating may occur in one spot, whereas other spots on the surface may be bare. Such products are acceptable in filtration processes due to the fact that the amount of impurities needed to be removed is relatively minor compared to the bulk liquid volume, and that uneven charge distributions can be compensated for by the depth of the filters. However, such products cannot readily be applied to delicate ion exchange processes. The number of active sites, as well as the accessibility of the active sites, are critical to the capacity of such processes. The chemical functional groups in ion exchangers cannot be buried close to the surface, but have to be somewhat removed from the surface, possibly with a molecular side arm for accessibility. One way of achieving this has been through the incorporation of particulates into a fibrous matrix modified by silanes which are chemically modified. Such silanes may carry functional groups such as DEAE, CM, or affinity chromatography sites. They are mechanically stable, strong and do not swell. However, they are expensive and show very high non-specific adsorption of protein by the silica hydroxy groups.
In sum, neither the affinity chromatography supports commonly used in laboratory-scale purifications nor the particulate- (or ion exchange modified particulate-) containing fibrous matrices for chromatography or filtration have proven to be of great use in scale-up of delicate affinity purification processes.
A need therefore continues to exist for supports useful in industrial-scale affinity chromatography purification processes, which will be noncompressible, controllably swellable, have high exchange capacity, exhibit high flow rates, be versatile, and be relatively inexpensive to produce, and for affinity matrices and devices comprising said supports.